Transposable elements and homotypic niches as drivers of immune dynamics and resistance in melanoma epigenetic-based immunotherapy.

9531 Background: Melanoma plasticity facilitates immune evasion and therapy resistance through dynamic cell-state transitions. While epigenetic remodeling critically influences these processes, its role in reshaping the tumor ecosystem under therapeutic pressure remains unresolved. We investigated the molecular and spatial mechanisms underlying clinical response to epigenetic-based immunotherapy in the phase Ib NIBIT-M4 trial (NCT02608437), which evaluated the DNA methyltransferase inhibitor guadecitabine combined with the anti-CTLA4 antibody ipilimumab. Methods: We performed longitudinal single-cell multiome profiling (paired snRNA-seq and snATAC-seq) on tumor biopsies collected at baseline and during treatment (weeks 4 and 12) from five melanoma patients (two responders, three non-responders). To resolve the spatial architecture of cell states, we integrated these data with high-resolution spatial transcriptomics (Visium HD) across nine samples. Analyses focused on defining malignant meta-programs (MPs), tumor microenvironment (TME) remodeling, and the regulatory role of transposable elements (TEs). Results: Integrated analysis resolved seven malignant MPs. Responders exhibited progressive enrichment of an Antigen Presentation/Interferon program, coupled by coordinated TME remodeling with T- and B-cell expansion. Conversely, non-responders were enriched for a de-differentiated Neural crest-like state. Spatial modeling revealed that this resistant Neural crest-like program relies on homotypic clustering to form compact, centrally localized niches that stabilize its transcriptional identity. In contrast, Wnt/β-catenin subpopulations displayed a bimodal architecture, existing as either cohesive clusters or dispersed plastic cells. Epigenetic therapy reactivated TEs, which provided regulatory signals priming innate immunity and generated tumor-associated antigens that drove immunoediting in responders. Mechanistically, NFATC2 emerged as a master regulator of the Neural crest-like phenotype, and its inhibition promoted re-differentiation toward immunogenic states. Conclusions: Epigenetic-based immunotherapy dynamically reshapes the melanoma ecosystem by modulating TE activity and cell-state plasticity. Clinical response links to TE-driven immune priming, whereas resistance is maintained by spatially consolidated Neural crest-like niches stabilized by NFATC2. These findings identify spatial clustering as a principle of resistance and highlight NFATC2 as a potential therapeutic target.